The objective of this proposal is to examine the formation of human centromeres, which ensure proper chromosome segregation during cell division. Errors in chromosome segregation lead to aneuploidy and mosaicism, resulting in birth defects and neoplasias. Normal human centromeres contain large amounts of repetitive alpha satellite DNA, which presents certain limitations to the analysis of their formation. However, neocentromeres, found on mitotically stable rearranged chromosomal fragments separated from normal centromeres, do not contain repetitive sequences and can be found localized to low or single copy genomic DNA. Thus, neocentromeres provide a novel approach to investigate current models that centromere formation requires either a distinct primary DNA sequence, e.g. alpha satellite or neocentromere DNA, or largely sequence independent epigenetic modifications, e.g. a distinct chromatin structure or temporal differences in DNA replication in DNA replication. Thus, the following three Specific Aims examine a unique collection of seven independent cell lines that each contain a supernumerary inversion/duplication 13q chromosome with a neocentromere. 1) Molecular cytogenetic FISH mapping will determine the positions of the neocentromeres and the inversion breakpoints to individual or overlapping cosmids from chromosome 13q. At least four neocentromeres in this collection have been cytogenetically localized to chromosome band 13q32. This FISH mapping will potentially define regions in 13q with a high propensity for neocentromere formation and/or a relationship to inversion breakpoints. 2) The role of primary DNA sequence in centromere formation will be addressed by isolation and sequence analysis of neocentromere DNA, using two complementary approaches. Modified extended chromatin techniques that retain the kinetochore protein CENP-C will be used to localize genomic clones to neocentromeres to high resolution. Neocentromere sequences will be isolated by immunoprecipitation of centromeric chromatin using antibodies to the centromere-specific histone CENP-1. 3) The role of DNA replication timing in centromere formation will be addressed by examination of the replication timing of neocentromere DNA and comparison to corresponding DNA sequences in homologous chromosomes, using two approaches. BrdU incorporation will permit assessment of replication of specific chromosomal regions in neocentromeric and non-centromeric states. In situ replication timing will permit assessment of the relative replication timing of specific genomic sequences at neocentromeres and on normal chromosomes.